Cryptate compounds and methods for diagnosis and therapy

ABSTRACT

The present invention relates to cryptate compounds useful as chelating agents. In particular, the present invention relates to functionalized derivatives of certain cryptate compounds. These functionalized derivatives are suitable for use in radiolabelling and similar applications. The present invention also relates to a method for diagnosis or therapy of a disease utilizing functionalized derivatives of cryptase compounds. In one embodiment, the present invention relates to a compound which is capable of being radiolabelled of general formula (I) in which n represents an integer from 2 to 4, where each R 4  and R 5  is independently selected from —H, CH 3 , COOH, NO 2 , CH 2 OH, H 2 PO 4 , HSO 3 , CN, C═ONH 2  and CHO; X and Y are the same or different and are selected from the group of C—R, N, P and C—Z in which R represents a hydrogen or halogen atom or a hydroxyl, nitro, nitroso, amino, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl or cyano group, or a group of the formula —COOR′, COCOOR′, NH—COCH 2 Br, —NH—CO—CH═CH—COOR′ in which R′ is a hydrogen atom or alkyl group; or, W is selected from the group of NH, S and O; and Z is a functionalized linkage group which is capable of binding said compound of formula (I) to a molecular recognition unit and wherein at least one of X and Y is C—Z; or a pharmaceutically acceptable salt thereof

TECHNICAL FIELD

The present invention relates to cryptate compounds useful as chelatingagents. In particular, the present invention relates to functionalisedderivatives of certain cryptate compounds. These functionalisedderivatives are suitable for use in radiolabelling and similarapplications. The present invention also relates to a method fordiagnosis or therapy of a disease utilising functionalised derivativesof cryptate compounds.

BACKGROUND OF THE INVENTION

Radiolabelled compounds are useful as radiopharmaceuticals, imagingagents, or the like which are especially useful for but not limited tothe diagnosis and therapy of diseases including cancer.

Known radiolabelled compounds suffer from the disadvantage that, in use,the radiolabelled nuclide can become detached from the carrier compoundthereby leading to problems and potential complications in diagnosticand therapeutic applications. Further, the known radiopharmaceuticalstend to be non-specific in their biodistribution throughout a subject.

The present invention seeks to ameliorate the stated disadvantages ofthe prior art by providing compounds which are capable of beingradiolabelled more expeditiously, specifically target a localised areaof tissue or an organ in a subject and which are more stable than theprior art compounds and less toxic. Further, the compounds of thepresent invention are typically suitable for use in pharmaceuticalformulations. It is a farther typical object of the present invention toprovide a method of diagnosis or therapy of disease in a subject.

SUMMARY OF THE INVENTION

In accordance with a first embodiment of the present invention, there isprovided a compound which is capable of being radiolabelled of generalformula (I) which is as follows:

in which n represents an integer from 2 to 4,where each R⁴ and R⁵ is independently selected from —H, CH₃, COOH, NO₂,CH₂OH, H₂PO₄, HSO₃, CN, C(═O)NH₂ and CHO;

-   -   X and Y are the same or different and are selected from the        group of C—R, N, P and C—Z in which R is selected from hydrogen,        halogen, hydroxyl, nitro, nitroso, amino, optionally substituted        alkyl, optionally substituted aryl, optionally substituted        aralkyl, cyano, —COOR′, COCOOR′, NH—COCH₂Br, —NH—CO—CH═CH—COOR′        in which R′ is a hydrogen atom or alkyl group;    -   W is selected from the group of NH, S and O; and    -   Z is a functionalised linkage group which is capable of binding        said compound of formula (I) to a molecular recognition unit and        wherein at least one of X and Y is C—Z; or a pharmaceutically        acceptable salt thereof.

It is to be understood that throughout this specification, the term“molecular recognition unit” includes an antibody, protein, peptide,carbohydrate, nucleic acid, oligonucleotide, oligosaccharide, liposome,or other molecule which can form part of a specific binding pair.

In one form of the compound of Formula (I), the functionalised linkagegroup Z is selected from the group of halogen or other leaving group,nitro, nitroso, imide, dione of the formula,

vinyl group of formula Het¹—C(Het²)═CH₂ (where Het¹ and Het² are thesame or different and is each a nitrogen containing heterocyclic groupor Het¹ is a nitrogen containing heterocyclic group and Het² is H),—C(═NH)OR², NCO, NCS, COR″, COOR′, SR², NHNR²R³, NHCONHNR²R³,NHCSNHNR²R³, CONR²R³, OR², NR²R³, (CH₂)_(p)R¹, (CH₂)_(p)ArR¹,(CH₂O)_(p)CH₂R¹, (CH₂OCH₂O)_(q)ArR¹, (CHCH)_(t)R¹, (CHCH)_(t)ArR¹ whereR² and R³ are the same or different and are independently selected fromH, (CH₂)_(p)R¹, (CH₂)_(p)ArR¹, (CH₂O)_(p)CH₂R¹, —(CH₂OCH₂O)_(q)ArR¹,(CHCH)_(t)R¹, (CHCH)_(t)ArR¹ and where R¹ is selected from SH, OH, NH₂,COOH, NCS, —N═N, or —C(═NH)OCH₃, COR″, where R″ is H, halogen, N₃,alkoxy, OAr, imidyloxy, imidazoyloxy, alkyl, or alkyl substituted with ahalogen or other leaving group, where p is an integer from 1 to 20, moretypically 1 to 10, still more typically 1 to 4, even more typically 1 to2 and yet more typically 1; q is an integer from 1 to 20, more typically1 to 10, still more typically 1 to 4, even more typically 1 to 2 and yetmore typically 1; r is an integer from 1 to 4, more typically 1 or 2,still more typically 1; and Ar is optionally substituted aryl oroptionally substituted aralkyl, provided that when one of X and Y isselected from C—NO₂, C—OH, C—Cl, C—CH₃ or C—NH₂ then the other X or Ysubstituent cannot be selected from C—NO₂, C—OH, C—Cl or C—NH₂. Inmoieties of formula (CH₂)_(p)R¹, (CH₂)_(p)ArR¹, one or more methylenegroups may also be replaced with O, S, NH or carbonyl, for exampleC(O)R¹, CH₂C(O)R¹, NHCH₂R¹, NHC(O)R¹, CH₂OR¹, CH₂C(O)NHR¹, and the like.

Typically in the compound of Formula (I), the functionalised linkagegroup Z of the compound of Formula (I) is selected from the group ofhalogen, maleimide, a vinyl pyridyl group of formula

or a dione of formula

NR²R³ where R² and R³ are the same or different and are independentlyselected from H, (CH₂)_(p)R¹, (CH₂)_(p)ArR¹, (CH₂O)_(p)CH₂R¹,—(CH₂OCH₂O)_(q)ArR¹, —(CHCH)_(r)R¹, (CHCH)_(r)ArR¹ and where R¹ isselected from NH₂, COOH, NCS, NCO, —N═N, or —C(═NH)OCH₃, COR″ groupwhere R″ is H, halogen, alkyl, or alkyl substituted with a halogen orother leaving group, where p is an integer from 1 to 20, more typically1 to 10, still more typically 1 to 4, even more typically 1 to 2 and yetmore typically 1; q is an integer from 1 to 20, more typically 1 to 10,still more typically 1 to 4, even more typically 1 to 2 and yet moretypically 1; r is an integer from 1 to 4, more typically 1 or 2, stillmore typically 1; and Ar is optionally substituted aryl or optionallysubstituted aralkyl, provided that at least one of R² and R³ is otherthan hydrogen, and wherein, in moieties of formula (CH₂)_(p)R¹,(CH₂)_(p)ArR¹, one or more methylene groups may also be replaced with O,S, NH or carbonyl, for example C(O)R₁, CH₂C(O)R¹, NHCH₂R¹, NHC(O)R¹,OR¹, SR¹, CH₂OR¹, CH₂C(O)NHR¹, and the like.

More typically in a compound of Formula (I), each R⁴ and R⁵ is H; W isNH; n is 2 to 4, more typically 2 or 3, still more typically 2; Z isselected from halogen and NR²R³ where R² and R³ are the same ordifferent and are independently selected from H, (CH₂)_(p)R¹,(CH₂)_(p)ArR¹, provided that at least one of R² and R³ is other than H;R¹ is selected from NH₂, COOH, NCS, NHCOCH₂Br and COR″ where R″ ishalogen, typically Br; and p is an integer from 1 to 4, more typically 1to 2 and still more typically 1. Yet more typically, R¹ is NH_(2.)Typically, R is amino, nitro, hydroxy or halogen and still moretypically R is amino.

As used herein, the term “aryl” refers to single, polynuclear,conjugated and fused residues of aromatic hydrocarbons or aromaticheterocyclic ring systems. Examples of such groups are phenyl, biphenyl,terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl,dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthracenyl,fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl, pyridyl,4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl, indolyl,pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolyl,isoquinolyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl,phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl, heteroaryl,pyridine and Het—CH═CH₂ and the like. Typically aryl is phenyl, pyridyl,naphthyl, anthracenyl or the like, and heteroaryl is typically pyridineand Het—CH═CH₂. Still more typically, aryl is phenyl.

As used herein, the term “aralkyl” refers to alkyl groups substitutedwith one or more aryl groups as previously defined. Examples of suchgroups are benzyl, 2-phenylethyl and 1-phenylethyl.

As used herein, the term “optionally substituted” means that the moietydescribed as optionally substituted may carry one or more substituentsselected from amino, halogen, hydroxy, mercapto, nitro, cyano,thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy,carboxyl, alkoxycarboxyl, carbamoyl, pyridoylamino,carboxyalkyl-carbamoyl, N-carboxyalkylcarbamoyl, sulpho, sulphamoyl,mono- or dialkylated or phenylated sulphamoyl which can carry one ormore alkyl substituents, alkylsulphonyl, alkoxysulphonyl, optionallyhydroxy-containing phenylsulphonyl or phenoxy sulphonyl.

In another form of the compound of Formula (I), the Z group of saidcompound of Formula (I) is selected from the group of NR²R³ where R² andR³ together with the nitrogen atom to which they are attached form asubstituted saturated or unsaturated 3 to 8 membered ring optionallycontaining one or more additional heteroatoms O, S or N and whereinthere is at least one substituent capable of binding said compound ofFormula (I) with a molecular recognition unit.

In accordance with a second embodiment of the present invention, thereis also provided a compound of Formula (I) as described in the firstembodiment of the present invention which is complexed with a metal ion.

The metal ion is typically selected from ⁶⁴Cu, ⁶⁷Cu, ^(99m)Tc, Ga, In,Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Mo, Ni, V, Pb, Ir, Pt, Zn, Cd,Mn, Ru, Pd, Hg, Ti, Tl, Sn, Zr, and the lanthanide group of elements inthe Periodic Table such as Sm, Ho, Gd, Tb, Sc, Y, and the actinides.

The metal ion is further typically a radionuclide selected from thegroup of ⁶⁴Cu ⁶⁷Cu, ^(99m)Tc, and radionuclides of In(III), Ga (III), Fe(III), Cu (II), Ti (IV) and other radionuclides from the Lanthanides,Re, Sm, Ho, and Y.

In accordance with a third embodiment of the present invention, there isalso provided a pharmaceutical formulation comprising a compound ofFormula (I) as described the first embodiment of the present inventionor a metal complex, radiolabelled complex or pharmaceutically acceptablesalt thereof together with a pharmaceutically acceptable carrier.

In accordance with a fourth embodiment of the present invention, thereis also provided a diagnostic formulation comprising a compound ofFormula (I) as described in the first embodiment of the presentinvention or a metal complex, radiolabelled complex or pharmaceuticallyacceptable salt thereof and a reducing agent in a pharmaceuticallyacceptable carrier.

In accordance with a fifth embodiment of the present invention, there isalso provided a method of diagnosis or therapy in a subject comprisingadministering to the subject a diagnostically or therapeuticallyeffective amount of a compound of Formula (I) as described in the firstembodiment of the present invention or a metal complex, radiolabelledcomplex or a pharmaceutically acceptable salt thereof.

In accordance with a sixth embodiment of the present invention, there isalso provided a use of a compound of Formula (I) or a metal complex,radiolabelled complex or pharmaceutically acceptable salt thereof in thepreparation of a medicament for diagnosis or therapy of disease in asubject.

In accordance with a seventh embodiment of the present invention, thereis also provided a compound of Formula (I) as described in the firstembodiment of the present invention or a metal complex, radiolabelledcomplex or pharmaceutically acceptable salt thereof when used in thediagnosis or therapy of disease in a subject.

In accordance with an eighth embodiment of the present invention, thereis also provided a conjugate compound comprising at least one compoundof Formula (I) as described in the first embodiment of the presentinvention or a metal complex, radiolabelled complex or apharmaceutically acceptable salt thereof bonded to at least onemolecular recognition unit comprising an antibody, protein, peptide,carbohydrate, oligonucleotide, oligosaccharide, liposome or the like.

In accordance with a ninth embodiment of the present invention, there isprovided a method of diagnosis or therapy in a subject comprisingadministering to the subject a diagnostically or therapeuticallyeffective amount of a conjugate compound as described in the eighthembodiment of the present invention.

In accordance with a tenth embodiment of the present invention, there isprovided a use of a conjugate compound as described in the eighthembodiment of the present invention in the preparation of a medicamentfor diagnosis or therapy of disease in a subject.

In accordance with an eleventh embodiment of the present invention,there is provided a conjugate compound as described in the eighthembodiment of the present invention when used in the diagnosis ortherapy of disease in a subject.

In accordance with a twelfth embodiment of the present invention, thereis provided ax method of imaging a subject comprising introducing acompound of Formula (I) or a metal complex, radiolabelled complex,conjugate compound or pharmaceutically acceptable salt thereof to asubject.

In accordance with a thirteenth embodiment of the present invention,there is provided a use of a compound of Formula (I) or a metal complex,radiolabelled complex, conjugate compound or pharmaceutically acceptablesalt thereof in the preparation of a medicament for imaging in asubject.

In accordance with a fourteenth embodiment of the present invention,there is provided a compound of Formula (I) or a metal complex,radiolabelled complex, conjugate compound or pharmaceutically acceptablesalt thereof when used in imaging in a subject.

In accordance with a fifteenth embodiment of the present invention,there is provided a compound of Formula (I) having the followingstructure:

DETAILED DESCRIPTION OF THE INVENTION

The compounds of Formula (I) typically comprise those compounds where nrepresents an integer from 2 to 4, more typically 2 or 3, still moretypically 2. Typically, one of X and Y is a C—Z group where Z istypically NR²R³ where R² and R³ are the same or different and areselected from H, (CH₂)_(p)R¹ and (CH₂)_(p)ArR¹, where R¹ is aspreviously defined, and p is 1 to 4, more typically 1 to 2 and stillmore typically 1, provided that at least one of R² and R³ is other thanhydrogen. Usually, one of X is a C—Z group and the other is a group C—R,where R is as previously defined, typically amino, lower alkyl, nitro,hydroxy or halogen. Generally, R¹ is NH₂ and p is 1.

An example of a compound of Formula (I) is:

The R¹ group in a compound of Formula (I) may provide a point ofattachment of a compound of Formula (I) to a molecular recognition unit.

The molecular recognition unit is typically an antibody, protein,peptide, oligonucleotide, oligosaccharide. In particular, the molecularrecognition unit is typically an antibody and more typically amonoclonal antibody.

Thus, compounds of Formula (I) provide a method of attachment ofradionuclide metal ions such as In(III), Ga(III), Fe(III), Tc(IV) orTc(V), Re(VII), Cu(II), Ti(IV), other radionuclides from theLanthanides, Rhenium, Samarium, Holmium, Yttrium and the like tomolecular recognition units such as monoclonal antibodies, receptorspecific proteins, peptides or oligonucleotides for in vivo imaging andtherapy.

The compounds of general Formula (I) are typically prepared byattachment of a functional linking group to a suitable cryptate. Methodsof synthesis of cryptates useful as precursors to compounds of Formula(I) in which W is NE are described in U.S. Pat. No. 4,497,737 in thename of Sargeson et al, the disclosure of which is incorporated hereinby reference. Other cryptates where W is S or O may be prepared byanalogous methods. Sargeson et al describe synthesis of metal cage“cryptate” compounds by the condensation of a tris-(diamine) metal ioncomplex as described at column 3 lines 30 to 35 with formaldehyde and anappropriate nucleophile. In order to obtain the compounds of Formula(I), an appropriate nucleophile is selected so as to obtain the desiredfunctionalised linkage group Z as defined in Formula (I). In particular,reference is made to column 4 lines 17 to 27 of U.S. Pat. No. 4,497,737.Alternatively, a functionalised linkage group Z may be attached to afunctional group of a cryptate prepared by the methods taught bySargeson et al (for example see Example 9 at column 9 line 65 to column12 line 10 of Sargeson et al) by standard synthetic techniques. Ifnecessary, a protecting group may be introduced into the cryptatestructure to protect latent functionality for the desired functionalisedlinkage group Z as defined in Formula (I) during synthesis of thedesired cryptate precursor. Suitable protecting groups are described,for example in Greene, T. W., Protective Groups in Organic Synthesis(John Wiley & Sons, New York, 1981) and McOmie, J. F. W., ProtectiveGroups in Organic Chemistry (Plenum Press, London, 1973).

For example, compounds of Formula (I) where said Z group comprises amono- or di-substituted amino group and where the substituent isoptionally substituted alkyl, are readily prepared by treating the aminocompound with the appropriate halo-substituted alkyl. Typically, thecompound of Formula (I) where Z is —NH—CH₂—CH₂—NH₂ can be prepared bytreating a compound of Formula (I) where R is NH₂ with BrCH₂CH₂NH₂ inthe presence of NaHCO₃ or the like with suitable protection.

Compounds of formula (I) where R¹ is —NCS may be prepared by reactingthe amino compound with thiophosgene (see WO87/12631), Kozak et al.,Cancer Res. 49, 2639 (1989). Substituted acid halide compounds areproduced by reacting a compound of formula (I) where R is NH₂ withBrCH₂COBr at 4° C. according to the procedure of C J Mathias et al.,Bioconjugate Chem., 1, 204 (1990). Compounds with an electrophilicmoiety can also be prepared by methods known in the art, such as in ACCChem. Res. 17 202-209 (1984). Compounds with active esters(CH)_(p)—C(O)—X may be formed by the procedures of Bodanszky M, ThePeptide. Analysis Synthesis Biology, Ed. E. Gross and J Meienhofer, Vol1, pp 105-196, Academic Press, Inc., Orlando, Fla. (1979) and BodanszkyM, Principles of Peptide Synthesis, pp 9-58, Springerverlag, N.Y.,(1984). Other compounds of formula (I) may be prepared from suchcompounds by standard procedures such as described in Modern SyntheticReactions, H O House, 2^(nd) Edition, Benjamin, Inc. Philippines, 1972.

In a typical embodiment, compounds of formula (I) in which X or Y is C—Zwhere the group Z is a group NH(CH₂)_(p)R¹ or NH(CH₂)_(p)ArR¹ may beprepared by a Schiff Base condensation reaction of a compound of formula(I) (or a metal complex thereof) in which X or Y is NH₂ with an aldehydeof formula HC(O)(CH₂)_(p−1)R¹ or HC(O)(CH₂)_(p−1)ArR¹. (Still typically,the Schiff Base condensation reaction is most appropriately conductedbetween a copper complex of compounds of Formula (I) in which X or Y isNH₂ with an aldehyde of formula HC(O)(CH₂)_(p−1)R¹ orHC(O)(CH₂)_(p−1)R¹.) In one particular form of this embodiment, acompound of formula (I) is obtained by the reaction of nitrobenzaldehydewith the copper complex of an aminocryptate such as described bySargeson et al. The reaction is typically performed in an inert-gasatmosphere and in the presence of solvent and diluents typically inertto the reactants. Suitable solvents comprise aliphatic, aromatic, orhalogenated hydrocarbons such as benzene, toluene, xylenes;chlorobenzene, chloroform, methylene chloride, ethylene chloride; ethersand ethereal compounds such as dialkyl ether, ethylene glycol mono or-dialkyl ether, THF, dioxane; alkanols such as methanol, ethanol,n-propanol, isopropanol; ketones such as acetone, methyl ethyl ketone;nitriles such as acetonitrile or 2-methoxypropionitrile; N,N-dialkylatedamides such as dimethylformamide; dimethylsulphoxide, tetramethylurea;as well as mixtures of these solvents with each other. If the amine or asalt thereof is soluble in water, then the reaction medium may be waterat low temperature. The compounds of formula (I) may be converted topharmaceutically acceptable salts by way of recognised procedures.

Typically, for medical use salts of the compounds of the presentinvention will be pharmaceutically acceptable salts; although othersalts may be used in the preparation of the inventive compound or of thepharmaceutically acceptable salt thereof. By pharmaceutically acceptablesalt is meant those salts which are, within the scope of sound medicaljudgement, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art.

Suitable pharmaceutically acceptable salts of the compounds of thepresent invention may be prepared by mixing a pharmaceuticallyacceptable acid such as hydrochloric acid, sulphuric acid,methanesulphonic acid, succinic acid, fumaric acid, maleic acid, benzoicacid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaricacid, or citric acid Suitable pharmaceutically acceptable salts of thecompounds of the present invention therefore include acid additionsalts.

For example, S. M. Berge el al. describe pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The saltscan be prepared in situ during the final isolation and purification ofthe compounds of the invention, or separately by reacting the tree basefunction with a suitable organic acid. Representative acid additionsalts include acetate, adipate, alginate, ascorbate, asparate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphersulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

The radiolabelling of compounds of formula (I), and salts thereof can beaccomplished by using procedures recognised in the art. For example,radiolabelling of the chelator with ⁶⁷Cu can be achieved by addingcopper in an aqueous acetate solution to a compound of formula (I) in anaqueous solution and incubating at room temperature.

Alternatively, the radiolabelling of a compound of formula (I) withtechnetium, for example, may be achieved by adding a reducing agent suchas stannous salts typically stannous chloride, to an aqueous solution ofa compound of formula (I), followed by reaction with aqueous sodiumpertechnetate solution (Na^(99m)TcO₄). The order of mixing rd thesethree components is believed not to be critical. However, typically thereducing agent is added to the chelator of formula (I). Other suitablereducing agents comprise alkali metal dithionites such as sodiumdithionite, sodium borohydride, hydrochloric acid, hydrobromic acid,other soluble dithionites such as potassium dithionite or ammoniumdithionite, a soluble bisulfite or metabisulfite such as sodiumbisulfite, potassium bisulfite, lithium bisulfite, ammonium bisulfite,sodium metabisulfite, potassium metabisulfite, lithium metabisulfite orammonium metabisulfite, or an aqueous solution of sulfur dioxide.

Technetium-99m in the form of an aqueous solution of sodiumpertechnetate is readily obtainable from commercially availablemolybdenum-99/technetium-99m generators or alternatively, instant^(99m)Tc may be used. Cu-64 is commercially available from AustralianNuclear Science & Technology Organisation and ⁶⁷Cu from the USDepartment of Energy, Brookhaven, USA.

The conjugate compounds of the eighth embodiment of this invention maybe formed by the reaction of a radiolabelled metal complex of a compoundof Formula (I) together with a molecular recognition unit. Theradionuclides which are useful for complexing with the compounds ofFormula (I) typically comprise metal ions which have at least twooxidation states, most typically an oxidation state of +2 or +3. In atypical embodiment, the radiolabelled metal complex is selected from⁶⁴Cu, ⁶⁷Cu and ^(99m)Tc, Sm, Ho, Re, Sc, Cu, Pd, Rh, and Y. The mosttypical metal ions comprise ⁶⁴Cu, ⁶⁷Cu, ^(99m)Tc. These radiolabelledmetal complexes are then reacted with a molecular recognition unit. Theradiolabelled molecular recognition unit so formed is useful fordiagnostic, therapeutic and radioimaging applications.

Alternatively a conjugate of a compound of formula (I) may be firstprepared, and then it may be radiolabelled.

Thus, radiolabelling of molecular, recognition units such asproteinaceous materials using compounds of formula (I) can be conductedin two ways, namely:

-   (a) prelabelling of a compound of Formula (I) with a suitable    radionuclide, followed by conjugation of the resultant    radiocomplexed compound to proteinaceous or other material, or-   (b) conjugating the compound of formula (I) to proteinaceous or    other material for subsequent radiolabelling.

The formation of a conjugate compound of formula (I) is usually achievedby the reaction of the functionalised linkage group with a thiol, amino,carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group presentin the molecular recognition unit. For example, an amino or hydroxygroup of the functionalised linkage group may be reacted with a freecarboxyl group of the molecular recognition unit, or vice versa.Suitably a coupling agent such as a carbodiimide may be employed tofacilitate the coupling reaction.

The conjugate compounds according to the eighth embodiment of thepresent invention may contain more than one molecule of a compound offormula (I) to any one molecular recognition unit. The metal complexingand radiolabelling of compounds of formula (I), and pharmaceuticallyacceptable salts thereof can be accomplished by using proceduresrecognised in the art. For example, the radiolabelling of the conjugatecompounds with ⁶⁴Cu may be achieved by adding an aqueous acetatesolution of ⁶⁴Cu to the conjugate compound in an aqueous solution andincubating for 5 minutes at room temperature. A composition comprisingan uncomplexed conjugate in accordance with the invention may also besupplied to radio-chemists, technicians, radiopharmacists, doctors orthe like in the form of a kit for radiolabelling immediately prior touse.

In a typical form of this invention, the kit comprises a first containerthat contains a radiolabelling metal ion, usually in solution, and asecond container that contains a conjugate compound as described in theeighth embodiment of the present invention. The kit, in use, theninvolves mixing the contents of said first and second containers toobtain the radiolabelled conjugate compound.

Typically, the compounds of Formula (I) or the metal complex,radiolabelled complex or pharmaceutically acceptable salt thereof areuseful for labelling molecular recognition units for use in methods ofdiagnosis and therapy of disease. In particular, the typicalradiolabelled molecular recognition units are monoclonal antibodies andfragments thereof, peptides, oligonucleotides, oligosaccharides orliposome or a part of a specific binding pair.

The applications of radiolabelled molecular recognition units comprisediagnosis, imaging and therapy of disease such as cancer. Typically, thecompounds of formula (I) and their metal complexes and/or salts thereofhave a diagnostic use as imaging agents in vitro and in vivo. The methodof diagnosis using the aforesaid imaging agents will result from thelocalisation of the radiolabelled conjugate compounds on specific organsand tissues in a subject.

The method of diagnosis will typically involve first the administrationof an effective amount of a radiolabelled compound of Formula (I) to asubject; and then monitoring the subject after a suitable period of timein order to ascertain the presence or absence of a cancer for example asevidenced by localisation of the radiolabel at a particular site in thesubject. Typically, the monitoring step shall provide informationregarding the location of any cancer if it is present. The effectiveamount or dosage of the radiolabelled compound of Formula (I) willdepend upon the desired amount of radioactivity required for thediagnostic application balanced with the safety requirement of notexposing the subject, in particular their organs and tissues, to harmfulamounts of radiation. Appropriate dosages for any given application maybe determined by persons skilled in the relevant art by no more thanroutine experimentation, given the teaching herein.

The method of therapy will typically involve compounds of formula (I) orthe metal complexes, radiolabelled complexes and/or pharmaceuticallyacceptable salts thereof which are useful as cytotoxic agents. In atypical embodiment, the therapy of disease comprises treatment ofcancer, abnormal cell disorders and the treatment of tumours. In suchapplications, the radiolabelled compound of formula (I) is typicallyconjugated to a molecular recognition unit which is capable of bindingspecifically to the tumour or abnormal cell. Examples of such molecularrecognition units comprise one part of specific binding pairs and areknown to persons skilled in the relevant art and typically compriseantibody/antigen pairs and the like.

The method of therapy will typically involve the administration of aneffective amount of a radiolabelled compound of Formula (I) to asubject. The effective amount or dosage will depend upon the desiredamount of radioactivity required for the diagnostic application balancedwith the safety requirement of not exposing the subject, in particulartheir organs and tissues, to harmful amounts of radiation. Appropriatedosages for any given application may be determined by persons skilledin the relevant art by no more than routine experimentation, given theteaching herein.

Typically the treatment would be for the duration of the condition, andcontact times would typically be for the duration of the condition.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages of a compound ofthe present invention will be determined by the nature and extent of thecondition being treated, the form, route and site of administration, andthe nature of the particular vertebrate being treated. Also, suchoptimum conditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thecompound of the present invention given per day for a defined number ofdays, can be ascertained by those skilled in the art using conventionalcourse of treatment determination tests.

Also included within the scope of the present invention are prodrugs ofthe inventive compound. Typically, prodrugs will be functionalderivatives of a compound of Formula (I) in accordance with the firstembodiment of the invention, which are readily converted in vivo to therequired compound for use in the present invention as described herein.Typical procedures for the selection and preparation of prodrugs areknown to those of skill in the art and are described, for instance, inH. Bundgaard (Ed), Design of Prodrugs, Elsevier, 1985.

When used in the treatment of disease, the compound of Formula (I) inaccordance with the first embodiment of the invention or a metalcomplex, radiolabelled complex or a pharmaceutically acceptable saltthereof, may be administered alone. However, it is generally preferablethat these compounds be administered in conjunction with otherchemotherapeutic treatments conventionally administered to patients fortreating disease. For example, a tumour may be treated conventionallywith surgery, and the compound of Formula (I) in accordance with thefirst embodiment of the invention or a metal complex, radiolabelledcomplex or a pharmaceutically acceptable salt thereof, to extend thedormancy of micrometastases and to stabilise and inhibit the growth ofany residual primary tumour.

Typically, when used in the treatment of solid tumours, compounds of thepresent invention may be administered with chemotherapeutic agents suchas: adriamycin, taxol, fluorouricil, melphalan, cisplatin, alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine,prednisone and procarbazine), vincristine, vinblastine, angioinhibins,TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609,SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like. Otherchemotherapeutic agents include alkylating agents such as nitrogenmustards including mechloethamine, melphan, chlorambucil,cyclophosphamide and ifosfamide; nitrosoureas including carmustine,lomustine, semustine and streptozocin; alkyl sulfonates includingbusulfan; triazines including dacarbazine; ethyenimines includingthiotepa and hexamethylmelamine; folic acid analogues includingmethotrexate; pyrimidine analogues including 5-fluorouracil, cytosinearabinoside; purine analogues including 6-mercaptopurine and6-thioguanine; antitumour antibiotics including actinomycin D; theanthracyclines including doxorubicin, bleomycin, mitomycin C andmethramycin; hormones and hormone antagonists including tamoxifen andcortiosteroids and miscellaneous agents including cisplatin andbrequinar.

When used in the treatment of disease, the compound of Formula (I) inaccordance with the first embodiment of the invention or a metalcomplex, radiolabelled complex or a pharmaceutically acceptable saltthereof, may be administered alone. However, it is generally preferablethat they be administered as pharmaceutical formulations. In generalpharmaceutical formulations of the present invention may be preparedaccording to methods which are known to those of ordinary skill in theart and accordingly may include a pharmaceutically acceptable carrier,diluent and/or adjuvant.

These formulations can be administered by standard routes. In general,the combinations may be administered by the topical, transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, oral, rectal or parenteral (e.g.intravenous, intraspinal, subcutaneous or intramuscular) route. Inaddition, the compound of Formula (I) in accordance with the firstembodiment of the invention or a metal complex, radiolabelled complex ora pharmaceutically acceptable salt thereof, may be incorporated intobiodegradable polymers allowing for sustained release, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of for example, a tumour or implanted so that theactive agents are slowly released systemically. Osmotic minipumps mayalso be used to provide controlled delivery of high concentrations ofthe active agents through cannulae to the site of interest such asdirectly into for example, a metastatic growth or into the vascularsupply to that tumour.

The carriers, diluents and adjuvants must be “acceptable” in terms ofbeing compatible with the other ingredients of the formulation, and notdeleterious to the recipient thereof.

Examples of pharmaceutically and veterinarily acceptable carriers ordiluents are demineralised or distilled water; saline solution;vegetable based oils such as peanut oil, safflower oil, olive oil,cottonseed oil, maize oil, sesame oils such as peanut oil, saffloweroil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil orcoconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;volatile silicones; mineral oils such as liquid paraffin, soft paraffinor squalane; cellulose derivatives such as methyl cellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and 1,2propylene glycol.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The radiolabelled molecular recognition units especially radiolabelledantibodies are particularly useful in medicine, for example, in locatingspecific tissue types and in the therapy of cell disorders. Theradiolabelled antibodies can also be used to target metal ions to aspecific tissue type, both in vitro and in vivo.

A typical use of the radiolabelled compounds of Formula (I): is toradiolabel monoclonal antibodies specific for colon, ovarian, lymphoma,breast and/or bladder cancer, with beta emitter radionuclides of metalssuch as Sm, Ho, Re, Sc, Cu, Pd, Pb, Rh and Y for therapy of abovementioned cancer. A further typical use is in the radiolabelling of amonoclonal antibody specific for metastasis of colon cancer fordiagnosis and therapy.

In another typical embodiment, the antibody in the conjugate compoundmay be a complete antibody molecule or a fragment thereof or an analogueof either of these, provided that the antibody comprises a specificbinding region. The antibody may be a humanised monoclonal or a fragmentthereof. The antibody may also be a recombinant antibody. The antibodymay be specific for any number of antigenic determinants, but istypically specific for one antigenic determinant.

In another typical embodiment, there is provided radiolabelling ofmonoclonal antibodies with ⁶⁷Cu (beta and gamma emitter) and ⁶⁴Cu(positron and beta emitter), for combined radioimmunoscintography (RIS)(SPECT and PET) and radioimmunotherapy (RIT). Other radionuclidescomprise Auger emitting agents where the compound of Formula (I) iscoupled to the monoclonal antibody and labelled with auger emittingisotope such as Fe-59 or Cu-64.

In still another typical embodiment, there is provided a two steppretargeted radioimmunotherapy where a monoclonal antibody with a firstmarker molecule attached thereto is injected into a subject. Once theantibody has cleared from the system and localised to the tumour, asecond injection is administered to the subject. This second injectiontypically involves the radiolabelled complex of Formula (I) attached toa second marker molecule which recognises the first marker molecule onthe targeted antibody. Alternatively, the second injection may typicallybe the second marker molecule alone and when cleared from the system,the radiolabelled complex of Formula (I) attached to the first markermolecule is administered to the subject. Both procedures provideamplification of the target site and reduce exposure of theradiolabelled complex to normal tissue. Still typically, the firstmarker molecule is biotin and the second marker molecule is avidin orstreptavidin. Still more typically, the first marker molecule is smallerthan the targeted antibody.

The invention also provides a two step procedure which involves theadministration of an antibody-DNA conjugate or antibody-oligonucleotideconjugate followed by targeting with a radiolabelled complementary DNAor complementary oligonucleotide. This procedure also providesamplification of the target site and reduces exposure complex of theradiolabelled to normal tissue.

The invention also provides a use of compounds of Formula (I) or metalcomplexes, radiolabelled complexes, conjugate compounds orpharmaceutically acceptable salts thereof as Magnetic Resonance Imaging(MRI) agents. In this form of the invention, there is typically formed acomplex of compound of formula (I) or a conjugate of the eighthembodiment, with a paramagnetic metal ion, typically Fe (III), Mn(II),which may be used as a contrast agent to enhance images. Further,complexes such as these may be employed in the form of a pharmaceuticalformulation where the complex is present with a pharmaceuticallyacceptable carrier, excipient or vehicle therefor.

The pharmaceutical formulations described for the different embodimentsof this invention typically comprise a formulation in the form of asuspension, solution or other suitable formulation. Physiologicallyacceptable suspending media together with or without adjuvants may beused. Still typically, the pharmaceutical formulations are in a liquidform and still more typically are in an injectable form. Still moretypically, the injectable formulations are dissolved in suitablephysiologically acceptable carriers which are recognised in the art.

The industrial use of the compounds of formula (I) further comprisestheir attachment to solid surfaces such as polymers, for use in theconcentration of metal ions and purification of water or attached to anelectrode surface for detection of specific metal ions.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further illustrated, by way of exampleonly, with reference to the accompanying figures; in which:

FIGS. 1 a and 1 b are graphical representations of the serum stabilityof ⁶⁷Cu complex of compound (4) at 0 and 7 days respectively;

FIG. 2 is a graphical representation of the effect of pH on thecomplexation of ⁶⁴Cu with compound (3);

FIG. 3 is a graphical representation of the biodistribution of a ⁶⁴Cucomplex of compound (4);

FIG. 4 is a graphical representation of the biodistribution of a ⁶⁴Cucomplex of compound (1),

FIG. 5 is a graphical representation of the biodistribution of a ⁶⁴Cucomplex of compound (3); and

FIG. 6 is a graphical representation of the biodistribution of ⁶⁴Culabelled conjugate of B72.3 conjugated with compound (3) in TumourBearing Nude Mice.

FIG. 7 is a graphical representation of the radiotherapeutic effect of30MBq of ⁶⁴Cu labelled conjugate of B72.3 conjugated with compound (3)in Tumour Bearing Nude Mice.

EXAMPLES

The following examples serve only to illustrate the invention and shouldnot be construed as limiting the generality of the above description.

Example 1 Preparation of Compound (3)

Preparation of compound (3) is illustrated in Schemes 1 and 2.

A. Preparation of Compound (2)

Compound (2) is prepared by Schiff base reaction of compound (1) withp-nitrobenzaldehyde. Synthesis of compound (1) is described in Aust. J.Chem (1994) 47, 143-179. A copper complex of (1) is dissolved in dryethanol (2.4 mmoles in 100 mls) and p-nitrobenzaldehyde (2.2 mmoles) isadded. The reaction mixture is evaporated to dryness and reconstituted(with dry ethanol) twice to remove any water. The reaction mixture isonce again reconstituted in dry ethanol and stirred for 30 mins undernitrogen gas. Sodium cyanoborohydride (25 mmoles), glacial acetic acid(2 mmoles) and activated 3 Å molecular sieves are added and the reactionis allowed to stir overnight. The mixture is filtered, evaporated todryness and extracted with chloroform and water (100 ml:200 ml). Thewater layer is diluted to 2 L, sorbed onto SP Sephadex C25 and elutedwith 0.3 M sodium acetate. (Scheme 1).

B. Preparation of Compound (3)

To palladium/C catalyst (20 mg) in water (0.5 ml) is added sodiumborohydride (50 mg) in water (0.5 ml) under nitrogen gas. To thismixture is then added compound (2) (30 mg) dissolved in approximately0.1 M sodium hydroxide (0.5 ml). The mixture is left to stir at roomtemperature for a further 30 mins or until the solution becomes clear. A2 ml glass vial is cooled on ice. The mixture is 0.22 μm filtered intothe cooled vial to remove the suspended palladium/C catalyst. To thiscooled filtrate is added concentrated hydrochloric acid dropwise untilall excess sodium borohydride is quenched (i.e. until gas evolution onaddition of acid ceases). The quantity of product is determined bytitration with a known concentration of ⁶⁴Cu as described in Example 2.Product is stored frozen at pH <1 in a rubber-stoppered vial undernitrogen gas. Yield: >95% (Scheme 2). The final product is characterisedby ¹H-NMR in D₂O at 298K (Bruker Avance DPX 400). ¹H NMR 3.28 ppm, m,6H, CH₂ (cage); 3.39 ppm, m, 6H, CH₂ (cage); 3.59 ppm, s, 6H, CH₂(cage); 3.71 ppm, s, 6H, CH₂ (cage); 4.45 ppm, s, 2H, CH₂; 7.51 ppm, d,2H, Ar; 7.70 ppm, d, 2H, Ar.

Example 2 Complexation of ⁶⁴Cu by Compound (3)

The effect of pH on complexation of ⁶⁴Cu by compound (3) wasinvestigated. Compound (3) was diluted into buffers of pH 3.0, 4.0, 5.0,6.0, 7.0, 8.0 and 9.0. A sufficient amount of ⁶⁴Cu was added and therate of complexation was monitored at (t=1, 2.5, 5, 10, 60 and 90 mins)(see method below) by Instant Thin Layer Chromatography (ITLC-SG).Complete complexation (>98%) was achieved within 1 min for all pH ≧4.0.The rate of complexation at pH 3.0 was slower. (FIG. 2).

Monitoring Complexation by ITLC-SG

ITLC-SG strips (10 cm×0.8 cm) were spotted with ˜1 μL of reactionmixture 1 cm from the bottom of the strip (origin) and were developed ina solvent containing 0.1 M sodium acetate (pH 4.5): ethanol=9:1.⁶⁴Cu-compound (3) remains at the origin (R_(f)=0.0), and “free” ⁶⁴Cuappears at the solvent front (R_(f)=1.0).

A typical method of radiolabelling the ligand is achieved by adjustingthe pH of an aqueous solution of the ligand to pH 5.0. Sufficient volumeof ^(64/67)Cu solution (usually in 0.02 M HCl or diluted into 0.1 Msodium acetate buffer pH 5.0) is added to form a 1:1 complex. Theefficacy of labelling is determined by ITLC-SG as described above. Onemain radiochemical species is observed.

Example 3 Serum Stability of ⁶⁷Cu Complex of Compound (4)

Serum stability studies were conducted by incubating a ⁶⁷Cu complex ofrelated species (4) in human plasma at 37° C.

At various time intervals, the complex was separated from the plasma bysize exclusion chromatography and the complex breakdown was assessed.Results indicated that no more than 2% of the copper is lost from thechelator during the first 174 hours at 37° C. (FIGS. 1 a and 1 b).

Example 4 Synthesis of B72.3 Conjugated with (3) Using1-Ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC)

A typical method for radiolabelling an antibody (such as B72.3) is toincubate the antibody with the ligand (such as compound (3)) in thepresence of EDC at pH 5.0 for 30 mins at 37° C. The unreacted ligand andEDC by-products are removed by washing with in buffer (pH 5.0) on a sizeexclusion ultra filtration membrane. The purified immunoconjugate isexposed to a slight excess of ^(64/67)Cu. The reaction is allowed toproceed at room temperature, and labelling is complete in less than 5mins. Excess ^(64/67)Cu is removed by washing with 0.1 M EDTA in PBS (pH7.2) on a size exclusion ultra filtration membrane or by separation on asize exclusion column (sephadex G25, eluted with PBS pH 7.2). (Scheme3).

The conditions for radiolabelling B72.3 were optimised over incubationtime (30 mins), reaction pH (5.0), concentration of B72.3 (5 mg/ml) andthe molar ratio of B72.3:EDC:ligand (1:1000:250).

Example 5 Biodistribution of Cu Complexes of (1), (3) and (4) in Balb/CMice

The biodistribution of the ⁶⁴Cu complexes of compounds (1), (3) and (4)(0.10 ml intravenous injection) were evaluated in balb/c mice (5 animalsper time point) at 3, 5, 10, 15, 20 and 30 minute time intervals.Biodistribution studies were performed in duplicate. Biodistribution ofthe radiolabel is presented in Tables 1, 2 and 3, and is illustrateddiagrammatically in FIGS. 3, 4 and 5.

Example 6 Evaluation of ⁶⁴Cu-Labelled Conjugate of B72.3 with Compound(3) in Tumour Bearing Nude Mice

The B72.3 antibody recognises the TAG-72 antigen which is expressed oncolorectal and ovarian tumours. The animal model used in the presentstudy uses LS174t cells which also express TAG-72 antigen. Thebiodistribution of the ⁶⁴Cu immunoconjugate (0.10 ml intravenousinjection) was evaluated in LS174t tumour bearing nude mice (5 animalsper time point) at 1, 3, 5, 24 and 48 hour time intervals.Biodistribution studies were performed in duplicate. Biodistribution ofthe radiolabel is presented in Table 4, and is illustrateddiagrammatically in FIG. 6 a; Biodistribution of complex of compounds(1) (2) and (3) show that if the ligand actually detaches from theantibody in any form it will clear rapidly from the system and notrelease the ⁶⁴Cu.

TABLE 1 ⁶⁴Cu-labelled compound (4) % INJECTED DOSE PER GRAM TIME 3 MIN 5MIN 10 MIN 15 MIN 20 MIN 30 MIN ORGAN MEAN S.D. MEAN S.D. MEAN S.D. MEANS.D. MEAN S.D. MEAN S.D. LIVER 1.81 0.30 1.59 0.12 1.16 0.06 0.64 0.065.01 0.55 5.76 0.58 SPLEEN 2.26 0.43 2.17 0.29 1.28 0.14 0.67 0.15 0.520.08 0.35 0.07 KIDNEY 20.08 4.98 18.62 4.09 14.79 6.26 7.52 0.54 25.993.24 30.26 4.50 MUSCLE 1.76 0.23 2.10 0.59 1.24 0.27 1.73 1.32 0.36 0.061.27 1.43 SKIN 5.98 0.31 6.46 0.56 4.21 0.68 2.76 0.10 1.73 0.45 1.420.42 BONE 1.60 0.25 1.56 0.24 1.28 0.23 1.60 0.41 0.89 0.51 0.65 0.10LUNGS 5.85 0.03 5.28 0.46 2.84 1.09 2.09 0.24 1.13 0.30 0.92 0.15 HEART3.25 0.34 2.66 0.14 1.60 0.36 1.08 0.18 0.51 0.08 0.47 0.17 BLOOD 8.250.32 7.08 0.22 4.41 0.78 2.71 0.57 1.40 0.12 1.07 0.21 BLADDER 13.156.81 28.05 18.33 69.91 54.03 8.17 4.96 16.38 20.77 25.38 35.78 STOMACH3.53 0.56 3.04 0.29 1.84 0.55 0.89 0.37 0.70 0.14 0.65 0.10 GIT 2.620.08 2.16 0.20 1.49 0.31 1.07 0.18 0.89 0.05 0.80 0.04

TABLE 2 ⁶⁴Cu-labelled compound (1) % INJECTED DOSE PER GRAM TIME 3 MIN 5MIN 10 MIN 15 MIN 20 MIN 30 MIN ORGAN MEAN S.D. MEAN S.D. MEAN S.D. MEANS.D. MEAN S.D. MEAN S.D. LIVER 2.29 0.22 1.50 0.17 1.42 0.22 0.82 0.190.69 0.15 0.50 0.08 SPLEEN 2.09 0.52 1.48 0.21 1.54 0.25 1.44 0.76 0.690.12 0.52 0.16 KIDNEY 23.18 11.73 11.50 1.34 12.80 1.83 6.77 1.40 7.770.67 4.95 0.92 MUSCLE 1.81 0.32 1.81 0.59 2.39 1.14 2.28 1.66 1.17 1.021.33 1.59 SKIN 5.82 0.42 5.59 0.59 5.48 1.07 2.61 0.44 2.96 0.72 1.630.08 BONE 2.25 0.45 1.87 0.47 1.79 0.29 1.23 0.12 1.25 0.44 0.78 0.23LUNGS 6.51 1.28 4.45 0.54 3.91 1.98 2.06 0.32 2.73 0.61 0.85 0.31 HEART3.11 0.38 2.25 0.10 2.27 0.43 0.89 0.12 1.23 0.71 0.40 0.10 BLOOD 8.681.35 6.32 0.70 5.45 0.81 2.48 0.31 2.63 0.29 1.14 0.25 BLADDER 24.3616.90 31.12 19.21 27.75 21.59 15.17 8.53 23.29 15.56 122.93 71.11STOMACH 3.50 0.64 2.73 0.36 2.08 0.80 1.50 0.46 1.16 0.06 0.74 0.04 GIT2.75 0.59 2.21 0.08 1.85 0.38 1.11 0.06 1.05 0.17 1.16 0.72

TABLE 3 ⁶⁴Cu-labelled compound (3) % INJECTED DOSE PER GRAM TIME 3 MIN 5MIN 10 MIN 15 MIN 20 MIN 30 MIN ORGAN MEAN S.D. MEAN S.D. MEAN S.D. MEANS.D. MEAN S.D. MEAN S.D. LIVER 1.94 0.17 2.00 0.52 1.46 0.39 1.24 0.231.04 0.08 0.72 0.02 SPLEEN 1.96 0.14 2.01 0.16 1.26 0.20 1.09 0.25 0.930.13 0.39 0.05 KIDNEY 22.41 5.77 18.99 2.98 13.41 3.04 10.26 1.76 9.981.73 5.95 0.27 MUSCLE 1.98 0.48 3.17 2.34 1.73 0.28 2.40 1.39 2.13 1.350.50 0.01 SKIN 5.63 1.05 6.18 1.17 4.86 1.14 4.49 1.03 3.41 1.10 1.450.13 BONE 1.68 0.04 2.11 0.60 1.29 0.43 1.36 0.24 1.11 0.08 0.72 0.10LUNGS 5.48 0.61 5.74 1.07 4.00 0.79 3.25 0.64 2.59 0.50 1.13 0.01 HEART2.79 0.36 3.00 0.59 2.03 0.58 1.56 0.29 1.24 0.05 0.50 0.11 BLOOD 7.740.43 8.19 1.53 5.23 1.28 4.39 0.59 3.05 0.44 1.25 0.02 BLADDER 11.364.02 12.31 4.09 14.36 9.79 10.54 9.64 13.45 16.47 2.74 2.02 STOMACH 2.690.51 3.11 0.79 2.35 0.65 1.91 0.45 1.54 0.33 0.60 0.04 GIT 2.66 0.292.88 0.46 1.80 0.38 2.92 2.46 1.05 0.22 0.55 0.03

TABLE 4 ⁶⁴Cu-labelled conjugate of B72.3 with compound (3) % INJECTEDDOSE PER GRAM TIME 1 HR 3 HR 5 HR 16 HR 24 HR 48 HR ORGAN MEAN S.D. MEANS.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. LIVER 19.09 1.05 18.44 0.5819.54 2.34 16.38 1.59 15.52 2.95 13.76 3.21 SPLEEN 11.51 1.13 8.35 1.1910.17 0.98 10.08 1.75 8.47 1.89 8.44 1.57 KIDNEY 10.64 1.47 9.94 0.1510.41 1.10 9.16 1.77 7.39 0.65 7.59 1.35 MUSCLE 1.45 0.52 1.48 0.23 1.450.16 1.98 0.27 2.54 0.54 1.72 0.42 SKIN 2.83 0.52 5.18 0.26 5.99 1.388.64 1.16 8.30 1.30 6.95 0.16 BONE 4.42 0.55 3.82 0.49 3.21 0.30 3.930.49 4.28 0.72 3.39 0.38 LUNGS 13.32 1.52 11.43 1.44 9.78 0.86 11.031.93 7.76 0.87 6.81 1.09 HEART 10.56 2.29 8.58 1.21 9.04 3.30 6.77 0.925.14 0.72 5.20 0.43 BLOOD 47.98 5.60 37.94 1.52 33.41 3.66 26.41 1.4821.50 1.81 18.69 2.01 BLADDER 2.33 0.77 2.93 0.62 3.15 0.47 7.00 5.095.41 2.37 4.87 0.92 STOMACH 1.41 0.29 1.69 0.33 2.32 0.51 1.48 0.74 1.580.31 1.43 0.32 GIT 2.23 0.35 2.60 0.20 2.87 0.23 2.28 0.14 2.20 0.201.86 0.31 TAIL 5.03 0.73 4.96 0.83 5.64 1.82 6.14 2.47 5.18 1.20 4.080.30 TUMOUR 5.98 0.80 11.10 0.76 12.31 1.68 23.77 2.46 29.43 4.44 38.434.79

TABLE 5 Biodistribution of ¹²³I-B72.3 in nu/nu mice % ID/g Time (hours)1 3 6 16 24 48 ORGAN Mean SD Mean SD Mean SD Mean SD Mean SD Mean SDLIVER 11.89 2.33 8.93 1.24 7.45 1.51 5.61 0.64 5.03 0.82 4.92 0.90SPLEEN 10.17 2.79 6.92 0.92 6.27 0.79 5.20 0.95 4.33 1.34 4.58 1.25KIDNEY 11.20 0.77 8.89 1.40 9.24 0.53 5.83 0.88 5.40 0.98 4.76 0.78MUSCLE 1.69 0.42 2.40 0.43 3.04 0.59 2.55 0.65 2.70 0.42 2.48 0.33 SKIN4.36 1.85 5.96 0.94 7.79 0.76 7.94 0.95 7.96 0.78 7.41 1.44 BONE 5.430.54 4.43 0.69 3.23 0.43 3.19 0.78 2.58 0.58 2.54 0.36 LUNGS 14.28 2.9712.84 2.89 11.00 1.57 7.39 1.62 6.64 1.57 6.47 0.64 HEART 14.03 6.2610.11 1.16 9.46 1.59 7.28 2.50 5.78 0.62 4.87 1.10 BLOOD 55.63 3.3644.78 4.03 42.20 3.93 28.27 3.00 28.73 2.23 22.73 2.50 URINE 3.96 0.5813.29 5.00 18.31 6.92 7.28 4.17 6.92 1.11 6.87 2.11 BLADDER 3.49 1.155.10 1.63 4.85 0.91 4.74 1.24 5.70 1.13 6.63 1.23 STOMACH 1.80 0.23 2.940.44 4.06 0.50 2.64 0.90 3.06 0.99 3.05 0.72 GIT 2.35 0.26 2.74 0.292.74 0.39 1.84 0.33 1.65 0.26 1.51 0.07 THYROID 8.76 3.60 17.29 6.2438.66 7.92 29.47 9.54 85.96 44.73 549.83 353.44 TUMOUR 9.61 2.04 16.461.61 31.43 8.39 31.37 6.54 44.67 8.50 46.17 7.25 % ID THYROID 0.08 0.020.19 0.03 0.42 0.11 0.40 0.18 1.24 0.64 2.59 1.13 TUMOUR:BLOOD 0.2 0.00.4 0.1 0.7 0.3 1.1 0.3 1.6 0.4 2.0 0.5 TUMOUR:LIVER 0.8 0.3 1.8 0.4 4.22.0 5.6 1.8 8.9 3.1 9.4 3.2 TUMOUR:KIDNEY 0.9 0.2 1.9 0.5 3.4 1.1 5.41.9 8.3 3.1 9.7 3.1 KIDNEY:BLOOD 0.2 0.0 0.2 0.0 0.2 0.0 0.2 0.1 0.2 0.00.2 0.1 KIDNEY:LIVER 0.9 0.2 1.0 0.3 1.2 0.3 1.0 0.3 1.1 0.4 1.0 0.3

TABLE 6 Biodistribution of ¹¹¹In-DTPA-B72.3 in nu/nu mice. % ID/g Time(hours) 1 3 6 16 24 48 ORGAN Mean SD Mean SD Mean SD Mean SD Mean SDMean SD LIVER 13.4 1.72 10.54 1.41 11.2 1.2 7.27 0.86 8.43 2.28 7.590.72 SPLEEN 12.52 2.66 9.56 1.96 10.05 2.55 6.47 3.33 7.32 1.43 4.482.28 KIDNEY 15.41 1.57 12.04 1.67 14.13 1.83 15.59 1.39 13.97 1.64 15.041.57 MUSCLE 2.08 0.81 1.96 0.42 2.56 0.32 2.44 0.82 2.28 0.26 1.84 0.51SKIN 4.17 1.98 7.54 1.53 9.31 1.41 9.48 1.21 8.44 0.42 7.54 0.75 BONE5.9 1.12 4.59 0.53 3.85 0.97 5.81 1.12 5.52 0.58 5.23 0.9 LUNGS 18.584.72 13.14 4.58 13.03 2.65 8.06 1.89 6.58 1.4 7.07 1.93 HEART 11.9 3.889.59 1.65 11.71 4.12 6.25 0.88 4.99 0.39 3.95 0.45 BLOOD 56.19 9.0439.03 4.66 38.23 3.64 26.51 1.72 21.78 2.36 17.04 0.89 URINE 5.23 0 3.961.16 4.46 1.68 4.46 1.12 4.43 1.56 BLADDER 11.82 9.4 5.59 2.85 8.3119.94 7.68 2.97 6.88 1.87 4.28 0.39 STOMACH 2.43 0.8 2.49 0.66 2.2 0.91.53 0.48 1.47 0.46 1.47 0.44 GIT 2.12 0.34 2.44 0.51 2.33 0.42 2.170.25 2.04 0.2 2.33 0.2 TAIL 9.37 5.68 8.39 4.38 8.53 5.27 4.98 1.17 4.690.96 3.79 1.37 THYROID 18.66 8.1 25.91 17.2 8.09 3.14 7.37 1.62 8.512.21 4.45 0.91 TUMOUR 8.87 2.39 17.95 4.93 21.24 6.11 38.25 8.71 35 5.2148.99 4 % ID THYROID 0.07 0.01 0.06 0.03 0.07 0.01 0.07 0.02 0.06 0.030.06 0.01 TUMOUR:BLOOD 0.2 0.1 0.5 0.2 0.6 0.2 1.4 0.4 1.6 0.4 2.9 0.4TUMOUR:LIVER 0.7 0.3 1.7 0.7 1.9 0.7 5.3 1.8 4.2 1.7 6.5 1.1TUMOUR:KIDNEY 0.6 0.2 1.5 0.6 1.5 0.6 2.5 0.8 2.5 0.7 3.3 0.6KIDNEY:BLOOD 0.3 0.1 0.3 0.1 0.4 0.1 0.6 0.1 0.6 0.1 0.9 0.1KIDNEY:LIVER 1.2 0.3 1.1 0.3 1.3 0.3 2.1 0.4 1.7 0.6 2.0 0.4It is to be understood that the term “SarAr” as used in the followingtables and description refers to compound (3) as shown in example 1.

TABLE 7 Maximum Radiation Dose Estimates for ⁶⁴Cu-SarAr-B72.3 Total DoseTotal Dose Organ mGy/MBq Rad/mCi Adrenals 0.030 0.111 Brain 0.018 0.068Breasts 0.021 0.078 Gallbladder Wall 0.036 0.133 LLI Wall 0.094 0.347Small Intestine 0.047 0.175 Stomach 0.029 0.107 ULI Wall 0.069 0.255Heart Wall 0.075 0.278 Kidneys 0.129 0.477 Liver 0.152 0.563 Lungs 0.0620.230 Ovaries 0.026 0.095 Muscle 0.013 0.047 Pancreas 0.030 0.110 RedMarrow 0.029 0.107 Bone Surfaces 0.030 0.111 Skin 0.019 0.069 Spleen0.088 0.327 Thymus 0.023 0.086 Testes 0.019 0.072 Thyroid 0.020 0.074Bladder Wall 0.019 0.070 Uterus 0.029 0.109 Total Body 0.024 0.090Tumour S factor Dose Dose Mass (g) rad/uCi-h Gy rad 0.1 2.25 17.801779.75 0.5 0.48 3.80 380.47 1.0 0.25 1.97 196.96 2.0 0.13 1.00 99.67

TABLE 8 Maximum Radiation Dose Estimate for ¹³¹I.B72.3 Total Dose TotalDose Organ mGy/MBq rad/mCi Adrenals 0.821 3.038 Brain 0.514 1.902Breasts 0.603 2.231 Gallbladder Wall 0.913 3.378 LLI Wall 2.24 8.288SmalI Intestine 1.3 4.810 Stomach 1.15 4.255 ULI Wall 1.69 6.253 HeartWall 1.82 6.734 Kidneys 2.41 8.917 Liver 1.92 7.104 Lungs 1.55 5.735Muscle 0.473 1.750 Ovaries 0.768 2.842 Pancreas 0.858 3.175 Red Marrow0.783 2.897 Bone Surfaces 0.678 2.509 Skin 0.547 2.024 Spleen 2.04 7.548Testes 0.595 2.202 Thymus 0.717 2.653 Thyroid 44.6 165.020 Bladder Wall0.755 2.794 Uterus 1.13 4.181 Total Body 0.715 2.646 Effective Dose 3.45mSv/MBq 12.765 rem/mCi (Tumour not included) Tumour Dose per mCi Mass(g) S factor Gy rad 0.1 3.6 982 98275 0.5 0.759 207 20720 1   0.393 10710728 2   0.2 54 5460Contribution to Organ Dose from Activity in TumourIt is assumed that the tumour is a small source located in the lowertrunk which will make a contribution to all other organ doses. So thatthe computer software program known as MIRDOSE3 can be used, theactivity is assumed to be located in the ovaries.The ovary dose given in above was calculated separately. It is not thetumour dose.Bladder Residence Time—The activity excreted via the bladder is almostinsignificant.The total bladder and urine residence times in Table 5 was used as theurine activity in the dose calculation. No excretion model or assumedvoiding time were used.

TABLE 9 Maximum Radiation Dose Estimates for ⁹⁰Y Total Dose Total DoseOrgan mGy/MBq rad/mCi Adrenals 0.57 2.109 Brain 0.57 2.109 Breasts 0.572.109 Gallbladder Wall 0.57 2.109 LLI Wall 3.47 12.839 Small Intestine1.36 5.032 Stomach 0.709 2.623 ULI Wall 2.25 8.325 Heart Wall 1.8 6.660Kidneys 7.87 29.119 Liver 2.89 10.693 Lungs 1.88 6.956 Muscle 0.2320.858 Ovaries 0.57 2.109 Pancreas 0.57 2.109 Red Marrow 0.964 3.567 BoneSurfaces 1.03 3.811 Skin 0.57 2.109 Spleen 2.32 8.584 Testes 0.57 2.109Thymus 0.57 2.109 Thyroid 1.72 6.364 Bladder Wall 0.444 1.643 Uterus0.57 2.109 Total Body 0.645 2.387 Tumour S Dose per mCi Mass (g)rad/uCi-h Gy rad 0.1 8.97 546 54585 0.5 2.51 152.7 15274 1   1.4 85.28519 2   0.758 46.1 4613Contribution to Organ Dose from Activity in TumourIt is assumed that the tumour is a small source located in the lowertrunk which will make a contribution to all other organ doses. So thatthe computer software program known as MIRDOSE3 can be used, theactivity is assumed to be located in the ovaries. The ovary dose givenin above was calculated separately. It is not the tumour dose.Bladder Residence Time: The activity excreted via the bladder is almostinsignificant.The total bladder and urine residence times in Table 5 was used as theurine activity in the dose calculation. No excretion model or assumedvoiding time were used.

TABLE 10 Radiation Dose Estimates for ¹³¹I-B72.3 (7 days) Total DoseTotal Dose Organ mGy/MBq rad/mCi Adrenals 0.377 1.395 Brain 0.24 0.888Breasts 0.276 1.021 Gallbladder Wall 0.418 1.547 LLI Wall 1.02 3.774Small Intestine 0.587 2.172 Stomach 0.52 1.924 ULI Wall 0.769 2.845Heart Wall 0.863 3.193 Kidneys 1.13 4.181 Liver 0.888 3.286 Lungs 0.7272.690 Muscle 0.212 0.784 Ovaries 0.35 1.295 Pancreas 0.393 1.454 RedMarrow 0.358 1.325 Bone Surfaces 0.309 1.143 Skin 0.25 0.925 Spleen0.951 3.519 Testes 0.272 1.006 Thymus 0.328 1.214 Thyroid 17.8 65.860Bladder Wall 0.338 1.251 Uterus 0.497 1.839 Total Body 0.323 1.195Tumour Dose to 24 hour Mass (g) rad/mCi 0.1 2991 0.5 631 1   327 2   166

TABLE 11 Radiation Dose Estimates for ⁹⁰Y (7 days) Total Dose Total DoseOrgan mGy/MBq rad/mCi Adrenals 0.5 1.850 Brain 0.365 1.351 Breasts 0.51.850 Gallbladder Wall 0.5 1.850 LLI Wall 2.87 10.619 Small Intestine1.14 4.218 Stomach 0.603 2.231 ULI Wall 1.87 6.919 Heart Wall 1.53 5.661Kidneys 6.5 24.050 Liver 2.41 8.917 Lungs 1.58 5.846 Muscle 0.194 0.718Ovaries 0.4 1.480 Pancreas 0.5 1.850 Red Marrow 0.808 2.990 BoneSurfaces 0.858 3.175 Skin 0.5 1.850 Spleen 1.97 7.289 Testes 0.5 1.850Thymus 0.5 1.850 Thyroid 1.43 5.291 Bladder Wall 0.38 1.406 Uterus 0.51.850 Total Body 0.546 2.020 Tumour rad/mCi at Mass (g) 24 hour 0.1 69250.5 1938 1   1081 2   585

Radiotherapeutic Study

Radiotherapeutic Study was conducted in two parts.

Part A where the theoretical doses to target and non-target organs werecalculated for the analogous radioimmunoconjugates.

Part B where the various radioactive levels of ⁶⁴Cu—SarAr—B72.3 wasinjected into tumour bearing mice and the therapeutic effect of theproduct was monitored as an extension of animal survival time.

Radiotherapeutic Study—Part A

Biodistribution studies of ¹²³I- and ¹¹¹In-radiolabelled B72.3 wereconducted in LS174t tumour bearing nude mice (see Table 5,6) Standardcalculations were performed using computer software MIRDOSE 3 which wasused to compare target to non-target dose of their analogous therapeuticcounterparts (⁹⁰Y and ¹³¹I respectively) with ⁶⁴Cu—SarAr—B72.3 (seeTable 7,8,9).

Theoretical maximum accumulated dose (which is equivalent to 10 halflife decay) for each radioimmunoconjugate was calculated. Total bodydose for ⁶⁴Cu—SarAr—B72.3 was significantly lower (0.09 rad/mCi) thananalogous products (¹³¹I—B72.3, 2.64 rad/mCi; Y—B72.3, 2.387 rad/mCi).Comparative maximum doses to tumours of various sizes was calculated.Doses for ¹³¹ I- and ⁹⁰Y—B72.3 appear to be better than for⁶⁴Cu—SarAr—B72.3. However, the lack of stability of theradioimmunoconjugate at the tumour site in real biological systemsprevents the tumour from receiving the maximum accumulated dose.

Hence doses to target and non-target organs were re-calculated assumingthe radioimmunoconjugates (¹³¹I and ⁹⁰Y) were stable for approximately24 hours at the tumour site and the non-target organs were dosed for upto 7 days allowing for natural biological clearance. MIRDOSE 3 was usedto re-calculate dose to various organs under these conditions (see Table10,11). Most relevant resultant target to non-target ratios are given inTable 12.

TABLE 12 Ratio of Target:Non-Target Doses for each Radioimmunoconjugate⁶⁴Cu ¹³¹I ⁹⁰Y Tumour (0.1 g):Kidneys 2094 716 288 Tumour (0.1 g):Liver1795 906 776 Total Body Dose for 7 days 0.09 1.195 2.020 *MIRDOSE 3 wasused to estimate human organ doses assuming the residence times in manare the same as the animal model. It is acknowledged that this affectsthe accuracy of the dose estimates.

Radiotherapeutic Study—Part B

In order to assess therapeutic effect of ⁶⁴Cu, in a real biologicalsystem, nude mice bearing LS174t colorectal carcinoma were injected withvarious doses (0, 10, 20, 30, 40 MBq) of ⁶⁴Cu—SarAr—B72.3.

Results are given in Table 13 and a typical profile of a study is givenin FIG. 1. For all activity levels greater than 20 MBq a significantextension in mouse life was achieved. Experimental details follow.

TABLE 13 Extension of mouse life relative to ⁶⁴Cu-SarAr-B72.3 - Part BExperiment Survival¹ (Days) Control² 20-25 10 MBq 30-35 20 MBq 60-70 30MBq 40-45 40 MBq 30-35 ¹Greater than 30% of animals; ²Greater than 50control animals;FIG. 1. Radiotherapeutic effect of 30 MBq of ⁶⁴Cu—SarAr—B72.3 in tumourbearing mice.

Experimental for Radiotherapeutic Study—Part B

-   Animal model: LS174t tumour tissue from nude mice was transplanted    into nude mice for each experiment.-   Animal selection: Only animals bearing tumours 3.5-5.5 mm    -   7 days post transplantation were selected for each study. Up to        10 animals per dose.-   Injection: The product was injected into the nude mice on Day 7    after transplantation of tumour tissue.-   Doses: Various activities of Cu-64-SarAr—B72.3 was injected into the    mice. Control animals receive only antibody.-   Monitoring of animals: Animals were monitored for changes in tumour    size    -   Animal mass    -   Behavioural and physical abnormalities    -   (e.g. movement/gait, food intake and hunching)    -   (For any of the above characteristic, frequency of weighing        increased)-   Histology, Haematology and Biochemistry:    -   Animals were sacrificed at pre-determined time points (2 days,        1, 2, 3, 4 weeks, 2, 3, 4, 5 and 6 months).-   Radiotoxic effects were monitored.    -   (conducted by external pathologist of Department of Veterinary        Anatomy and Pathology, University of Sydney)-   Endpoint of the study:    -   1) Body weight loss >20%.    -   2) Rapid weight loss of >10% overnight.    -   3) Ulceration of tumour    -   4) Limitation of normal behaviour (e.g. ability to feed or        drink).    -   5) Tumour size 10×10 mm (UK Cancer Council)

Example 7

In accordance with the description of the invention provided abovespecific preferred pharmaceutical compositions of the present inventionmay be prepared, and examples of which are provided below. The followingspecific formulations are to be construed as merely illustrativeexamples of formulations and not as a limitation of the scope of thepresent invention in any way.

A compound of Formula (I) may be administered alone, although it ispreferable that it be administered as a pharmaceutical formulation.

Example 7(a) Composition for Parenteral Administration

A pharmaceutical composition of the present invention for intramuscularinjection could be prepared to contain 1 mL sterile isotonic saline, and1 mg of compound of Formula (I).

Similarly, a pharmaceutical composition for intravenous infusion maycomprise 250 ml of sterile Ringer's solution, and 5 mg of compound ofFormula (I).

Example 7(b) Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection may be prepared by mixing 1% by weight ofcompound of Formula (I) in 12% by volume propylene glycol and isotonicsaline. The solution is sterilised by filtration.

Modifications and variations such as would be apparent to a skilledaddressee are deemed to be within the scope of the present invention. Itis to be understood that the present invention should not be limited tothe particular embodiment(s) described above. Throughout thespecification, unless the context clearly indicates otherwise, the word,“comprise”, “comprises”, “comprising” or other variations thereof shallbe understood as meaning that the stated integer is included and doesnot exclude other integers from being present even though those otherintegers are not explicitly stated.

Further, the present invention relates to all steps, compounds,intermediates as well as final products.

1. A compound which is capable of being radiolabelled of general Formula(I):

in which n represents an integer from 2 to 4, where each R⁴ and R⁵ isindependently selected from —H, CH₃, COOH, NO₂, CH₂OH, H₂PO₄, HSO₃, CN,C(═O)NH₂ and CHO; X and Y are the same or different and are selectedfrom the group consisting of C—R, N, P and C—Z in which R is selectedfrom hydrogen, halogen, hydroxyl, nitro, nitroso, amino, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaralkyl, cyano, —COOR′, COCOOR′, NH—COCH₂Br, —NH—CO—CH═CH—COOR′ in whichR′ is a hydrogen atom or alkyl group, wherein at least one of X and Y isC—Z; W is selected from the group of NH, S and O; and Z is afunctionalised vinyl pyridyl group which is capable of binding saidcompound of formula (I) to a molecular recognition unit, selected from

or a pharmaceutically acceptable salt thereof.
 2. A compound having thefollowing structure:

wherein said compound is capable of binding to a molecular recognitionunit.
 3. A compound according to claim 2 which is complexed with a metalion.
 4. A compound according to claim 2 which is complexed with a metalion selected from the group consisting of Cu, Tc, Gd, Ga, In, Co, Re,Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg,and Ti.
 5. A compound according to claim 4, wherein the metal ion is aradionuclide selected from the group consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In,Gd, Ga, Fe, Co, Ti and Re.
 6. A compound according to claim 5, whereinthe radionuclide is selected from ⁶⁴Cu and ⁶⁷Cu.
 7. A methodradioimaging comprising administering to a subject an effective amountof a radiolabelled metal ion complex of a compound, wherein saidcompound has the structure

or a pharmaceutically acceptable salt thereof.
 8. The method of claim 7,wherein said metal ion is selected from the group consisting of Cu, Tc,Gd, Ga, In, Y Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt, Zn,Cd, Mn, Ru, Pd, Hg, and Ti.
 9. The method of claim 8, wherein the metalion is a radionuclide selected from the group consisting of ⁶⁴Cu, ⁶⁷Cu,Tc, In, Gd, Ga, Fe, Co, Ti, and Re.
 10. The method of claim 9, whereinthe radionuclide is selected from ⁶⁴Cu and ⁶⁷Cu.
 11. A compoundaccording to claim 1, wherein the molecular recognition unit is selectedfrom the group consisting of an antibody, protein, peptide,carbohydrate, nucleic acid, oligonucleotide, oligosaccharide andliposome.
 12. A compound according to claim 1, wherein W is NH.
 13. Acompound according to claim 1, wherein said compound is complexed with ametal ion selected from the group consisting of Cu, Tc, Ga, In, Co, Re,Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg,Ti, Y and Sc.
 14. A compound according to claim 1, wherein n is 3 or 4and the compound is complexed with a metal ion is selected from thelanthanide group of elements in the Periodic Table.
 15. A compoundaccording to claim 13, wherein the metal ion is selected from the groupconsisting of Cu, Tc, Gd, Ga, In, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni,V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg, Ti, and Sc.
 16. A compound accordingto claim 13, wherein the metal ion is a radionuclide selected from thegroup consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In, Ga, Fe, Co, Ti, Re, and Y.
 17. Acompound according to claim 16, wherein the radionuclide is selectedfrom ⁶⁴Cu and ⁶⁷Cu.
 18. A pharmaceutical composition, for radioimagingcomprising a compound of Formula (I) compound according to claim 1, apharmaceutically acceptable salt thereof, or a radiolabelled complexthereof, together with a pharmaceutically acceptable carrier.
 19. Adiagnostic composition for radiodiagnosis comprising a radiolabelledcomplex of a compound of Formula (I) according to claim 1, or apharmaceutically acceptable salt thereof, and a reducing agent, togetherwith a pharmaceutically acceptable carrier.
 20. A method of diagnosingcancer comprising administering to a subject an effective amount of aradiolabelled complex of a compound of Formula (I) according to claim 1,or a pharmaceutically acceptable salt thereof, and determining whetheror not said subject has the cancer.
 21. The method of claim 20, whereinsaid metal ion is a radionuclide selected from the group consisting ofCu, Tc, Ga, In, Y, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir,Zn, Cd, Mn, Ru, Pd, Hg, Ti, and Sc.
 22. The method of claim 21, whereinthe metal ion is a radionuclide selected from the group consisting of⁶⁴Cu, ⁶⁷Cu, Tc, In, Ga, Fe, Co, Ti, Re, and Y.
 23. The method of claim22, wherein the radionuclide is selected from ⁶⁴Cu and ⁶⁷Cu.
 24. Amethod of radioimaging a subject comprising administering to saidsubject an effective amount of a radiolabelled metal ion complex of acompound of Formula (I) according to claim 1, or a pharmaceuticallyacceptable salt thereof.
 25. A compound according to claim 4, whereinthe metal ion is selected from the group consisting of Cu, Tc, Ga, In,Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Zn, Cd, Mn, Ru, Pd, Hg,and Ti.
 26. A pharmaceutical composition comprising a compound accordingto claim 2, or a pharmaceutically acceptable salt thereof, a metal ioncomplex thereof, or a radiolabelled complex thereof, together with apharmaceutically acceptable carrier.
 27. A diagnostic compositioncomprising a radiolabelled metal ion complex of a compound according toclaim 2, or a pharmaceutically acceptable salt thereof, and a reducingagent, together with a pharmaceutically acceptable carrier.
 28. Acompound according to claim 2, wherein the molecular recognition unit isselected from the group consisting of an antibody, protein, peptide,carbohydrate, nucleic acid, oligonucleotide, oligosaccharide andliposome.
 29. A compound according to claim 28, wherein the molecularrecognition unit is an antibody.
 30. A conjugate compound comprising atleast one compound of Formula (I) according to claim 1, or a metalcomplex, or radiolabelled complex, or a pharmaceutically acceptable saltthereof, bonded to at least one molecular recognition unit comprising anantibody, protein, peptide, carbohydrate, oligonucleotide oroligosaccharide.
 31. A conjugate compound comprising a compoundaccording to claim 2, or a metal complex, or radiolabelled complex, or apharmaceutically acceptable salt thereof, bonded to at least onemolecular recognition unit comprising an antibody, protein, peptide,carbohydrate, oligonucleotide or oligosaccharide.
 32. A compoundaccording to claim 31, wherein the molecular recognition unit is anantibody.
 33. A method of radioimaging a subject comprisingadministering to said subject an effective amount of a radiolabelledmetal ion complex of a conjugate compound according to claim 30, or apharmaceutically acceptable salt thereof.
 34. A method of radioimaging asubject comprising administering to said subject an effective amount ofa radiolabelled metal ion complex of a conjugate compound according toclaim 31, or a pharmaceutically acceptable salt thereof.
 35. Apharmaceutical composition comprising a conjugate compound or a metalion complex thereof according to claim 30, together with apharmaceutically acceptable carrier.
 36. A pharmaceutical compositioncomprising a conjugate compound or a metal ion complex thereof accordingto claim 31, together with a pharmaceutically acceptable carrier.
 37. Amethod of radiotherapy of cancer comprising administering to a subjectan effective amount of a radiolabelled metal ion complex of a compound,wherein said compound has the structure

or a pharmaceutically acceptable salt thereof.
 38. The method of claim37, wherein said radiolabelled metal ion is a radionuclide selected fromthe group consisting of Cu, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi, Cr,W, Ni, V, Ir, Zn, Cd, Mn, Ru, Pd, Hg, and Ti.
 39. The method of claim38, wherein the metal ion is a radionuclide selected from the groupconsisting of ⁶⁴Cu, ⁶⁷Cu, In, Ga, Fe, Co, Ti, and Re.
 40. The method ofclaim 39, wherein the radionuclide is selected from ⁶⁴Cu and ⁶⁷Cu. 41.The method of claim 24, wherein said metal ion is a radionuclideselected from the group consisting of Cu, Tc, Ga, In, Y, Co, Re, Fe, Au,Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Zn, Cd, Mn, Ru, Pd, Hg, Ti, Lu,and Sc.
 42. The method of claim 41, wherein said radionuclide isselected from the group consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In, Ga, Fe, Co,Ti, Re, Lu and Y.
 43. The method of claim 42, wherein the radionuclideis selected from ⁶⁴Cu and ⁶⁷Cu.
 44. A method of radiotherapy of adisease comprising administering to a subject an effective amount of aradiolabelled metal ion complex of a compound of Formula (I) accordingto claim 1, or a pharmaceutically acceptable salt thereof.
 45. Themethod of claim 44, wherein said metal ion is a radionuclide selectedfrom the group consisting of Cu, Tc, Ga, In, Y, Co, Re, Fe, Au, Ag, Rh,Pt, Bi, Cr, W, Ni, V, Pb, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg, Ti, Sc, Sm andLu.
 46. The method of claim 45, wherein the metal ion is a radionuclideselected from the group consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In, Ga, Fe, Co,Ti, Re, Sm, Lu and Y.
 47. The method of clam 46, wherein theradionuclide is selected from ⁶⁴Cu and ⁶⁷Cu.
 48. The method according toclaim 44, wherein n is 3 or 4 and the compound is complexed with aradiolabelled metal ion selected from the lanthanide group of elementsin the Periodic Table.
 49. A method of radiotherapy of cancer comprisingadministering to a subject an effective amount of a radiolabelled metalion complex of a conjugate compound according to claim 30, orpharmaceutically acceptable salt thereof.
 50. A method of radiotherapyof cancer comprising administering to a subject an effective amount of aradiolabelled metal ion complex of a conjugate compound according toclaim 31, or pharmaceutically acceptable salt thereof.
 51. A method ofradioimaging cancer comprising administering to a subject an effectiveamount of a radiolabelled complex of a compound of Formula (I) accordingto claim 2, or a pharmaceutically acceptable salt thereof.
 52. Themethod of claim 51, wherein said metal ion is a radionuclide selectedfrom the group consisting of Cu, Tc, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt,Bi, Cr, W, Ni, V, Ir, Zn, Cd, Mn, Ru, Pd, Hg, and Ti.
 53. The method ofclaim 52, wherein the metal ion is a radionuclide selected from thegroup consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In, Ga, Fe, Co, Ti, and Re.
 54. Themethod of claim 53, wherein the radionuclide is selected from ⁶⁴Cu and⁶⁷Cu.
 55. A method of diagnosing cancer comprising administering to asubject an effective amount of a radiolabelled complex of a compound ofFormula (I) according to claim 2, or a pharmaceutically acceptable saltthereof, and determining whether or not the subject has cancer.
 56. Amethod of radiotherapy of cancer comprising administering to a subjectan effective amount of a radiolabelled metal ion complex of a compoundof Formula (I) according to claim 1, or a pharmaceutically acceptablesalt thereof.
 57. The method of claim 56, wherein said metal ion is aradionuclide selected from the group consisting of Cu, Ga, In, Y, Co,Re, Fe, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Pb, Ir, Pt, Zn, Cd, Mn, Ru,Pd, Hg, Ti, Sc.
 58. The method of claim 57, wherein the metal ion is aradionuclide selected from the group consisting of ⁶⁴Cu, ⁶⁷Cu, Tc, In,Ga, Fe, Co, Ti, Re, Sm, Lu and Y.
 59. The method of claim 58, whereinthe radionuclide is selected from ⁶⁴Cu and ⁶⁷Cu.